Toxicity mechanisms of aflatoxin M1 assisted with molecular docking and the toxicity-limiting role of trans-resveratrol

In this study, AFM1 toxicity and the protective role of trans-resveratrol (t-rsv) against this toxicity were investigated with the help of multiple parameters in albino mice. As a result, AFM1 (16 mg/kg b.w) administration caused a decrease in body, kidney and liver weights. This reduction was associated with a decrease in feed consumption. AFM1 induced an increase in AST and ALT enzyme parameters and BUN, creatinine and MDA levels and a decrease in GSH levels. These increases have been associated with liver and kidney cell damage. AFM1 decreased MI and encouraged increases in MN and CAs numbers. The decrease in MI was correlated with AFM1-tubulin and the increase in CAs was associated with the AFM1-DNA interaction, which was demonstrated by molecular docking and spectral shifting. Besides, the decrease in DNA damage and amount was demonstrated by the comet assay technique. Administration of t-rsv (10 and 20 mg/kg b.w) reduced the toxic effects of AFM1 and caused a dose-dependent improvement in all physiological, biochemical and cytogenetic parameter values studied. For this reason, foods containing t-rsv or food supplements should be consumed in the daily diet to reduce the effect of toxic agents.


Material and methods
Test materials and experiment protocol. In this study, 36 healthy male Mus musculus (12-14 weeks old, 25-30 g) were used as subjects and obtained from GRU-Experimental Animals Laboratory. Albino mice were maintained in stainless steel cages, at 22 ± 3 °C and 55 ± 5% relative humidity, 12 h light/12 h dark cycle. All experiments were performed in accordance with the guidelines of the Animal Experiments Local Ethics Committee of Giresun University and approved by the Animal Ethics Committee of Giresun University (protocol number: 2017/02). This study was carried out in compliance with the ARRIVE guidelines. Six groups were formed with six mice in each group. The groups and the application to which the group is exposed are given in Fig. 1.
In the literature, the LD 50 value for AFM 1 has been estimated in the range of 9-16 mg/kg, and the upper limit of 16 mg/kg was preferred in this study 13 . In the selection of t-rsv dose, the dose range in which resveratrol provides protection against chemicals in mice was preferred 14 . Mice were brought to the laboratory where the experimental stages would be conducted seven days ago to adapt to the environmental conditions. The water, thane anesthesia were rinsed with distilled water, and epithelial cells were taken from the left and right buccal mucosa and spread on the slide. Preparations fixed in methanol: acetic acid solution were stained with Fast Green and Feulgen then examined with a research microscope. For the MN test in erythrocytes, blood samples (5 µL) from the tail veins of mice stunned with halothane anesthesia were mixed with EDTA (3%) solution and spread on sterile slides. The preparations were fixed in ethanol (70%) for 2 min. were left to dry at 21 °C for 24 h. Slides were stained with Giemsa (5%) for 15 min. and analyzed under a microscope. For the leukocyte MN test, blood samples were obtained from each mouse and centrifuged at 5000 rpm for 10 min. 5 mL of 0.075 M KCl solution was transferred to the pellet. After incubation for 20 min., the solution was centrifuged at 5000 rpm for 10 min. and a washing solution consisting of 3:1 methanol/acetic acid was added and the mixture incubated at − 20 °C for 30 min. then leukocyte cells were spread and examined under a microscope after staining with Giemsa 4 .
CA and MI analysis. CAs and MI analysis were determined in the bone marrow. For this aim, mice treated with 0.025% colchicine were sacrificed 2 h later under halothane anesthesia. Bone marrow obtained from the femurs of mice was aspirated, washed with the physiological solution and 0.075 M KCl was transferred. After fixation with Carnoy's solution, samples stained with Giemsa (5%) were examined under a microscope 18 . MI rates and CAs frequencies were determined in the prepared slides and 1.000 cells were analyzed for each group. In prepared slides, MI was determined as the percentage of dividing cells among 1000 nucleated cells for each group. Recovery effects (RE) of t-rsv against AFM 1 induced genotoxicity were calculated by using Eq. (1). In determining RE, data belonging to Group VI, where t-rsv provided the highest healing effect, Group IV, in which AFM 1 was treated alone, and the control group were used. D 1 : data of AFM1 + t-rsv treated group, D 2 : Data of AFM 1 treated group, D 3 : data of control.
Comet assay (single-cell gel electrophoresis). The protocol of Tice et al. 19 was performed for alkaline single cell gel electrophoresis with slight modifications. Slides were dipped in 1% normal melting point agarose for coating and allowed to dry at 37 °C. 10 µL of peripheral blood were added to 120 µL of 0.5% low-meltingpoint agarose at 37 °C, layered onto a coated slide, covered with a coverslip and left at 4 °C for 5 min. to solidify the agarose. The coverslip was removed and the slides were immersed into a lysis solution (2.5 M NaCl, 100 mm Na 2 EDTA, 10 mM Tris-HCl buffer, pH 10, 1% Triton X-100) for approximately 1 h. After lysis, the slides were transferred to a horizontal gel electrophoresis tank with a fresh and cooled alkaline buffer. After a 20 min. DNA unwinding period, electrophoresed at 0.86 V/cm (20 V, 300 mA) for 20 min. Slides were stained using ethidium bromide staining solution after carefully flushing three times with Tris-buffer (0.4 M Tris, pH 7.5) for 5 min. The www.nature.com/scientificreports/ preparations were washed with cold water to remove excess stain and covered with a coverslip. To prevent DNA damage, all steps were performed in low light and analyzed by fluorescence microscopy. Comets were analyzed with Comet Assay software version 1.2.3b 20 with the parameters of tail DNA length. A total of 600 cells were analyzed for each group, 100 in each animal for DNA damage. The extent of DNA damage was scored from 0 to 4 depending upon the level of DNA damage. The cells were classified into five categories based on tail DNA length ranging from zero to four according to Collins 21 . The total DNA damage per group, expressed as arbitrary units, was calculated using Eq. (2).

Molecular docking.
Molecular docking studies were carried out to elucidate the mechanism of the cytotoxic and genotoxic effects of AFM 1 . For this purpose, potential interactions of AFM 1 with DNA molecules, histone and tubulin proteins were investigated. The cyro-em 3D structure of tubulin (alpha-1B chain and tubulin beta chain) (6RZB) 22 , the crystal 3D structure of histone proteins (histone H3.1, histone H4, histone H2A and histone H2B type 1-A) (3X1T) 23  The DNA solution was prepared by gentle shaking in 0.01 M sodium nitrate solution. DNA-AFM 1 interaction was evaluated by investigating the change in absorbance of mixtures containing DNA and different concentrations of AFM 1 (1:1, 1:2, 1:4). The UV absorption spectrum of DNA-AFM 1 complex in the range of 220-300 nm was obtained 31 . UV absorption spectra were recorded on the Mapada UV-6100PCS double beam spectrophotometers.
Statistical analysis. Statistical analysis of the data obtained from experimental stages was carried out using the SPSS for Windows V 22.0 (SPSS Inc, Chicago, IL, USA) package program. One-way ANOVA and Duncan tests were used to evaluate the statistical differences between the experimental groups, respectively. Obtained data were shown as mean ± SD and were considered statistically significant when p values were < 0.05. Pearson correlation analysis (two-sided) was performed in RStudio and correlation plots were performed with the corrplot package 32 . Principal component analysis (PCA) was performed for physiological, biochemical and genetic parameters, which are different biomarkers of toxicity for each dose tested. The FactoMineR 33 and factoextra 34

Results and discussion
Alterations in body and organ weights. The effects of AFM 1 and t-rsv on the body and organ weights in albino mice are given in Table 1. Body weights of Groups I, II and III increased significantly at the end of the application period. There was no statistically significant difference between these groups in terms of body weight gain (p > 0.05) and an increase in body weight in the range of 12.4-11.62 g was recorded for these three groups. Body weight, liver and kidney weights decreased 17.5%, 43.1% and 51.3%, respectively, in the AFM 1 applied group compared to the control group. These reductions are directly related to the feed intake of the mice. Feed consumption of each group was followed for 28 days. There was no statistically significant change in feed consumption for 28 days in Groups I, II and III. In the AFM 1 treated group, feed consumption was found to be similar on the 7th and 14th days, but decreased significantly on the 21st and 28th days compared to control. The adverse effects of AFM 1 application on body and organ weights are directly related to the reduction in feed intake as well as indirectly related to impaired protein/lipid metabolism, anorexia, inhibition of lipogenesis and protein synthesis. Lipogenesis-lipolysis balance has an important role in increasing body weight and especially lipogenesis induces weight gain 36 . Lipid metabolism abnormalities, which may occur as a result of toxic effects of aflatoxins on the liver and other tissues, significantly affect the weight gain and organ weights of organisms.
Although there is no study on the effect of AFM 1 on weight gain and feed intake in albino mice, there are important data with other aflatoxin types. Arvind and Churchil 37 reported weight gain of chickens fed with AFB 1 was reduced 33.94% compared to control. Dimitri et al. 38 determined that AFB 1 and AFG 2 treatments caused a body weight loss of approximately 539 g in rabbits compared to the control group, and this loss was associated with disruptions in protein and DNA synthesis. Hussain et al. 39 determined that AFB 1 administration caused depression, decrease in feed intake, body weight and defecation in rats. Contrary to our findings, Casado et al. 40 stated that AFB 1 administration did not cause any change in feed consumption and weight gain in mice. Treatment of t-rsv with 16 mg/kg b.w. AFM 1 resulted in an improvement in body weight and organ weights compared to Group IV. While 3.5 g weight gain was recorded at the end of the 28th day in AFM 1 treated group, an increase of 5.62 g and 7 g in body weights were detected in Groups V and VI in which t-rsv and AFM 1 were administered together. Although these increases lagged behind the control group, a significant improvement was achieved compared to the only-AFM 1 treated group. The positive effects of t-rsv on body weight gain were also observed in organ weights. In Group VI treated with t-rsv and AFM 1 , liver and kidney weights increased by 30.5% and 40.3%, respectively, compared to the AFM 1 treated group. Similar increases were observed in feed intake, and feed consumption increased significantly in Group V and Group VI compared to the AFM 1 -treated group. T-rsv administration provided significant protection against a decrease in body and organ weights, and this protection was statistically significant especially at 20 mg/kg dose compared to the permethrin-only group (p < 0.05). This curative property of t-rsv on weight gain and feed consumption can be explained by the suppression of the toxic effects exhibited by AFM 1 . Resveratrol has an important effect on lipid metabolism in organisms. Resveratrol prevents oxidative stress-induced LDL oxidation and lipid peroxidation. It also plays an important role in lipid metabolism by decreasing low-density lipoprotein and total cholesterol levels and increasing plasma high-density lipoprotein levels. The regulatory role of resveratrol in lipid metabolism and its activity to repair damage in liver and kidney tissues are the most important factors of recovery in body and organ weights 4 . In the literature, it has been reported that resveratrol application improves the changes in body and liver weights observed in organisms under the influence of various exogenous factors 41,42 . Antioxidant and oxidant dynamics. In order to investigate the effects of AFM 1 and t-rsv applications on antioxidant/oxidant balance in liver and kidney tissues, GSH and MDA levels were measured and the results are given in Fig. 2. MDA and GSH levels were found to be similar in liver and kidney tissues in the control group and only t-rsv treated groups. Abnormal increases in MDA levels were detected in the group treated with 16 mg/ kg AFM 1 . MDA levels of the liver and kidney increased 1.22 and 1.41 times, respectively, in the AFM 1 applied    43,44 . Similarly, Shen et al. 45 reported that AFB 1 administration induces lipid peroxidation and causes cell damage in rat liver cells. The increase in the level of oxidant molecules in the cells causes a decrease in the levels of endogenous antioxidants and the deterioration of the antioxidant/oxidant balance. The significant decrease in GSH levels in the liver and kidney tissues in AFM 1 treated group confirms this hypothesis. 16 mg/kg AFM 1 application decreased the GSH levels in the liver and kidney by 63.7% and 39.3%, respectively, compared to the control group. GSH, which has a tripeptide structure, is a non-enzymatic antioxidant and provides neutralization of free radicals in cells. In cells, glutathione can be found in two different forms: reduced (GSH) and oxidized (GSSG). The balance and ratio between reduced glutathione and oxidized glutathione in cells are used to evaluate the cellular oxidative damage 46,47 . In healthy cells and tissues, more than 90% of the total glutathione is in reduced form. The decrease in the reduced glutathione level indicates the presence of oxidative stress in the cell and the deterioration of the antioxidant/oxidant balance. As a result, increased MDA and decreased GSH levels after AFM 1 treatment in the liver and kidney confirm that AFM 1 is an important inducer of oxidative stress and disrupts the antioxidant/oxidant balance. Within the scope of antioxidant/oxidant dynamic analysis, it was determined that t-rsv administration caused an improvement in antioxidant/oxidant balance, which was impaired by AFM 1 . AFM 1 + 20 mg/kg t-rsv administration provided 34.4% and 22.8% improvement in liver and kidney GSH levels compared to the AFM 1 -treated group. The dose-dependent increase in GSH level and 16% decrease in MDA level are indications that t-rsv provides an improvement in the antioxidant/oxidant balance in the liver and kidney. Resveratrol reduces oxidative stress in the cell by different mechanisms and protects cellular structures. Some of these mechanisms are direct suppression of free radicals, induction of the activity of antioxidant enzymes such as glutathione peroxidase, superoxide dismutase and catalase. Resveratrol also reduces lipid peroxidation by neutralizing free radicals and preserves cell membrane integrity 48 . All these effects of resveratrol reduced the deterioration in antioxidant/ oxidant dynamics induced by AFM 1 . While there is no result in the literature regarding the effect of t-rsv against oxidative damage induced by AFM 1 , it has been reported to be protective against oxidative damage caused by various chemicals. Şener et al. 49 reported that administration of 30 mg/kg resveratrol in rats regulated the impaired antioxidant balance and caused an improvement in the decreased GSH level. and AST enzyme activities, which are indicators of liver cell damage, creatinine and BUN, which are accepted as indicators of kidney cell damage, were investigated. No difference was observed in terms of serum parameters examined in Groups I, II and III. This result shows that t-rsv administered alone did not cause a statistically significant difference in serum parameters. There were significant increases in all tested parameters in Group IV, which was administered 16 mg/kg AFM 1 . After AFM 1 administration, AST, ALT, BUN and creatinine levels increased by 33.6%, 35.8%, 43.6% and 58.4%, respectively, compared to the control group. These results show that AFM 1 administration causes damage to liver and kidney tissues, especially the damage to kidney tissue occurs at a higher rate. AST is found in many tissues such as the liver, lung, kidney, brain, heart, pancreas and skeletal muscle. AST is an intracellular enzyme and its serum levels are quite low. After damage occurs in the tissues where AST is present, it passes from the damaged tissue cells to the blood, and the serum level also increases. Although a high AST level in the serum is an indicator of tissue damage, it is not sufficient for the detection of liver damage alone. ALT is concentrated in the liver and is therefore considered a direct indicator of liver damage 50 . It is known that aflatoxin species cause hepatocellular necrosis, inhibition of polymerase activity, biochemical and pathological changes in liver cells 51 . As a result of these abnormalities caused by aflatoxins, cell damage occurs, enzymes leak into the blood and their levels increase. The increase in both AST and ALT levels together in this study is an indication that AFM 1 exposure induces liver damage. Although there is no study in the literature investigating the effects of AFM 1 on the liver or liver markers, other types of aflatoxins are reported to have similar effects. Han et al. 52 reported that administration of 20-40 μg/kg AFB 1 caused liver damage and significant increases in serum ALT and AST levels. In this study, similar to increases in AST and ALT, increases in BUN and creatinine levels were also observed after 20 µg/kg AFM 1 . Creatinine, which is formed as a breakdown product of creatine phosphate in muscle tissue, is excreted from the body by the kidneys. The amount of nitrogen in urea formed as a result of protein catabolism is expressed as BUN. Urea formed in the liver is removed from the body through the kidneys in the urine. It is known that creatinine and BUN levels increase in conditions such as kidney diseases, obstruction of the urinary tract and kidney stones 53 . Aflatoxins cause toxic effects in kidney tissue such as damage to glomerular capillaries, occlusion of cortical blood vessels, inflammation, coagulation necrosis, focal bleeding and occlusion areas 54 . These damages cause an increase in BUN and creatinine in the blood. The significant increases in BUN and creatinine levels observed in this study indicate that 16 mg/kg b.w. AFM 1 exposure causes renal damage. Eraslan et al. 55 reported that administration of 500 μg/kg aflatoxin in albino rats induced kidney damage, resulting in significant increases in BUN, creatinine and uric acid levels. www.nature.com/scientificreports/ In this study, it was also determined that abnormal increases in serum parameters regressed with AFM 1 + t-rsv administration. This regression indicates the protective property of t-rsv and this protection increases depending on the dose. AST, ALT, BUN and creatinine levels in Group V administered with 10 mg/kg t-rsv + AFM 1 decreased by 11.2%, 10.7%, 14.9% and 20.5%, respectively, compared to AFM 1 -treated group. Same improvements in 20 mg/kg t-rsv + AFM 1 treated group (Group VI) were 22.5%, 21.6%, 26.2% and 34.2%, respectively. As the t-rsv dose increased, the improvements in serum parameters also increased and the differences between Group V and Group VI were statistically significant in terms of each serum parameter (p < 0.05). The improvements in serum parameter levels after t-rsv administration proves the protective effect of t-rsv on liver and kidney tissue. The results of the antioxidant and oxidant dynamics analysis of this study revealed that AFM 1 causes oxidative stress. Oxidative stress-induced by AFM 1 causes significant damage to liver and kidney tissues. Resveratrol provides protection by preventing the oxidation of macromolecules in the liver and kidney 56 . Although there is no study in the literature on the healing effects of t-rsv against AFM 1 -induced damage, it is reported that resveratrol decreases damage in the liver and kidney induced by chemical agents. Akosman et al. 57 reported that 40 mg/kg resveratrol administration provided significant protection against liver and kidney injuries.
Analysis of genotoxic effects. The genotoxic effects of AFM 1 and the protective role of t-rsv were investigated by MN and CAs analyses. MN formations were investigated in leukocyte, erythrocyte and buccal mucosa cells, CAs were investigated in bone marrow cells. The effects of AFM 1 and t-rsv applications on MN frequency are given in Fig. 4. While no MN formation was observed in buccal epithelial cells, statistically insignificant MN formation was detected in leukocyte cells in control and only-t-rsv applied groups (p > 0.05). Negligible levels of MN were observed in the erythrocyte cells of the control group and 10 mg/kg t-rsv treated group (p > 0.05). This similarity between t-rsv-treated groups and the control group indicates that t-rsv does not have an inducing effect on MN formation. Significant levels of MN formation were detected in the erythrocyte, buccal epithelium and leukocyte cells of the AFM 1 -applied group. Among the cells, the highest MN formation was observed in leukocyte cells and the lowest in buccal epithelial cells. MN formation in a cell indicates the genotoxic effects and the presence of an agent that induces this effect. MNs can arise from single-stranded and double-stranded DNA breaks or lagging chromosomes. A nuclear membrane forms around these formations and MNs appear, similar in structure to the main nucleus, stained in the same color but smaller in size 58 . The formation of MNs is due to aneugenic or clastogenic effects, and the size of the MN also provides information about the type of these effects. Aneugenic agents cause centromere division errors, defects on spindle apparatus and lagging chromosomes, resulting in larger MN formations. Clastogenic agents cause DNA chain breaks, acentric fragments or chromosome breaks, resulting in the formation of MN in smaller sizes 59 . While the presence of MN indicates a genotoxic effect in the cell, the size of MN provides information about the mechanism of this effect. AFM 1 exposure induced large MN in leukocyte and erythrocyte cells and smaller MN in buccal mucosa cells. This result shows that AFM 1 exhibits both aneugenic and clastogenic effects by causing spindle apparatus defects, centromere division errors, acentric fragments or chromosome breaks. Similarly, Corcuera et al. 60 reported that AFB 1

administration induced MN formation in bone marrow cells in rats.
The CAs types observed in Group IV-treated with AFM 1 confirm that AFM 1 has both aneugenic and clastogenic effects. The effects of AFM 1 and t-rsv on CAs frequency in bone marrow cells are given in Table 2. No statistically significant CAs formation was found in the control group and only t-rsv administered groups. Negligible fragment formation was observed only in the 10 mg/kg t-rsv treated group. AFM 1 application caused significant CAs formations with high frequency. Chromosome breaks are the most common type of CAs and fragment, gap, ring, acentric and dicentric chromosome abnormalities are also other types of CAs induced by AFM 1 . Both MN formations and the observation of different types of CAs indicate the genotoxic effects of AFM 1 . The size of MNs induced by AFM 1 provides information about the mechanism of toxicity; also the types of CAs give information about the genotoxicity mechanisms of AFM 1 . The high frequency of break, ring and fragment formation in bone marrow cells indicates the clastogenic effect of AFM 1 . As a result of the clastogenic effect, DNA chain breaks or chromosome breaks occur and these abnormalities cause the formation of other CA types. Chromosomes recombine at their breaking points and turn into ring chromosomes. Ring chromosomes that cannot be pulled to the poles in mitosis cause chromosome loss or excess in cells, resulting in high MN frequency 61 . Acentric and dicentric chromosomes are formed as a result of chromosome breaks. A dicentric chromosome is an abnormal chromosome with two centromeres and is formed by the fusion of segments originating from two chromosome breaks containing a centromere. As a result of the fusion of broken chromosome parts, dicentric chromosomes may occur, as well as acentric parts without a centromere 62 . When CA types induced by AFM 1 are examined, it can be said that a high frequency of chromosome breaks is induced and other CA types are formed as a result of the re-arrangement of chromosomal breaks. While no study on the genotoxicity of AFM 1 in albino mice has been reported in the literature, there are studies investigating the effects of other aflatoxin species. Fetaih et al. 63 reported that AFB 1 administration causes macro-DNA damages such as gaps, breaks, deletions, dicentric chromosomes, adherent chromosomes, hypopolyploidy, centromeric rearrangements in rats.
Another important result obtained from genotoxicity studies is that t-rsv treatment has a dose-dependent protective role in reducing the genotoxic effects. 10 mg/kg and 20 mg/kg t-rsv treatment with AFM 1 resulted in a 22.6-63.9% reduction in CA types. The most significant reduction was observed in gap formation, and 20 mg/ kg t-rsv application reduced gap formation by 63.9%. Considering that other CA types are generally thought to originate from chromosomal breaks, the decrease in break frequencies caused a decrease in other CA types as well. This genotoxicity-limiting effects of t-rsv can be associated with its antioxidant activity. Resveratrol is a molecule that neutralizes free radicals and potently induces intracellular antioxidants such as glutathione and catalase. Resveratrol, with its regulatory role on the cell cycle, suppresses the division of damaged cells and provides an opportunity for the cell to remain in the G 0 /G 1 phase and repair the damage. In this way, it provides   Although there is no study in the literature investigating the protective feature of t-rsv against AFM 1 toxicity, its protective feature against genotoxicity has been demonstrated by many studies. Carsten et al. 66 reported a significant reduction in the rate of radiation-induced chromosomal damage after resveratrol administration in mouse bone marrow cells.
DNA fragmentation. The effects of AFM 1 and t-rsv on DNA fragmentation were investigated by Comet assay. DNA fragmentation in the comet assay was evaluated on the basis of DNA damage score. Comet analysis data of AFM 1 and t-rsv applied groups are given in Fig. 5. T-rsv application alone did not induce DNA damage, and no statistically significant difference (p > 0.05) was found between the control group (Group I) and the t-rsv alone treatment groups (Group II and III). AFM 1 treatment was induced DNA damage in leukocyte cell nuclei of swiss albino mice, as evidenced by the results. While the average DNA damage score in Group I (control group) was 11.17 ± 0.79, the average DNA damage score in Group IV, which was treated with 16 mg/kg AFM 1 , was 255.67 ± 16.57. T-rsv treatment in addition to AFM 1 had a dose-dependent protective effect. DNA damage score was 170.50 ± 14.26 in Group V administered with 16 mg/kg AFM 1 + 10 mg/kg t-rsv and 133.83 ± 11.43 in Group VI administered 16 mg/kg AFM 1 + 20 mg/kg t-rsv. The results demonstrated that AFM 1 treatment-induced DNA fragmentation, but t-rsv treatment had a dose-dependent protective effect. There are statistically significant differences in DNA damage scores between Groups I-III and IV-VI (p < 0.05).

Molecular docking and spectral measurements of AFM 1 -DNA interactions. As a result of CAs
analysis, AFM 1 application caused abnormalities in the chromosome structures. Molecular docking of AFM 1 and histone proteins were performed to predict the possible mechanism of AFM 1 genotoxicity. Histones are proteins that allow DNA to condense properly into chromosomes. Multiple hydrogen bonds, hydrophobic interactions and ionic bonds allow histones to bind to DNA. These bonds are usually formed between the amino acid backbones of histones and the sugar-phosphate backbone of DNA. Weakening of these bonds between histone proteins and DNA or deformations in the histone structure cause instability in the genome structure. Modifications in histone proteins affect different processes in the cell such as chromosome packaging, DNA damage and DNA repair 67 . Molecular docking analysis including inhibition coefficients and the binding energy of AFM 1 and histone proteins are given in Fig. 6 and Table 3. Histone H3.1 and AFM 1 interacted with hydrogen bonding via amino acid Thr59 and hydrophobic interactions via amino acids Val62, Leu63 and Leu97. This interaction occurred with a binding energy of − 5.30 kcal/mol and an inhibition constant of 131.29 µM. In the interaction between Histone H4 and AFM1, hydrogen-bonding interactions and hydrophobic interactions occurred with a binding energy of − 5.43 kcal/mol and inhibition constant of 105.48 µM. Histone H2A and AFM 1 interacted by hydrogen bonds with amino acid residues Arg72 and Gln76 and hydrophobic interactions with amino acid residues Arg72, Ala75, Leu82, Arg83 and Phe84. AFM 1 interacted with Histone H2B type 1-A by hydrogen bonding with Lys59 and hydrophobic interaction with amino acid residues Ile26, Leu62, Lys59, Ile66, Ile29 and Leu58. AFM 1 -DNA interactions were investigated by molecular docking and spectral measurements. Molecular docking of AFM 1 -DNA and the binding constants are given in Fig. 7 and Table 4. AFM 1 had contact with B-DNA dodecamer (1BNA) with − 8.08 kcal/mol binding energy and an inhibition constant of 1.20 µM. AFM 1 exhibited The results of molecular docking studies involving AFM 1 and DNA molecules revealed that AFM 1 has the ability to connect with DNA molecules, particularly at nucleotides in the same strand. The binding of AFM 1 to A-A-T, A-C, A-A, G-G-C-C-C-C nucleotides can cause conformational changes in the structure of DNA. DNA-AFM 1 molecular docking also indicates the intercalation potential of AFM 1 . Intercalation causes reductions in helix winding of DNA conformation and alters the supercoiling structure. Intercalator-induced helix relaxation can cause DNA conformational changes, DNA bending and disruption of its integrity. This conformational change on DNA is not only localized at the intercalation sites but can also proceed along the DNA chain. Intercalating agents can also cause single-strand breaks in DNA, which can occur at the intercalation point or a forward point. These breaks usually occur in the form of proteinassociated DNA breaks. These proteins can be DNA repair enzymes or DNAase enzymes that cause breaks in DNA. Intercalators also have mutagenic properties and can cause many gene/chromosome mutations as well as break formations 68 . AFM 1 's intercalator feature can cause structural changes in DNA, disruption of helical structure and integrity. This intercalator potential of AFM 1 may also explain the formation of CAs. Aflatoxins exhibit genotoxic effects by many mechanisms. In addition to an indirect genotoxic effect by inducing the formation of oxidative stress, they can also create a direct genotoxic effect by interacting with DNA. The interactions of AFB 1 with different DNA sequences and histone fractions have been investigated by various methods in the literature. It has been reported in the literature that AFB 1 interacts with histone F2b and histone F 1 69 . Stark et al. 70 and Loechhler et al. 71 reported that AFB 1 binds to DNA and acts as an intercalator. In this study, it was determined that AFM 1 interacts with histone H3.1, H4, H2a and H2b fractions by forming hydrogen bonds and hydrophobic interactions. In addition, AFM 1 showed the ability to bind with DNA sequences, especially through nucleotides in the same chain. Briefly, AFM 1 exhibited a similar mechanism to AFB 1 , interacting with the tested DNA sequences and histone proteins, and acting as an intercalator. The interaction of AFM 1 and DNA, shown by molecular docking, was also supported by the UV absorption spectrum and the results are given in Fig. 8. The addition of AFM 1 to the DNA solution caused hypsochromic and hyperchromic shifts in the  Table 3. Potential molecular interactions and binding affinities of AFM 1 with histone proteins. www.nature.com/scientificreports/ UV spectrum. The hypsochromic shift was from 260 nm to approximately 240 nm, and the hyperchromic shift was from 1.176 to 1.202. As the AFM 1 ratio increased, the shift intensity also increased. The hypsochromic shift confirms the AFM 1 -DNA interaction. Spectral hyperchromicity of DNA (increase in absorbance) also indicates partial instability of the secondary structure of DNA resulting from the interaction 72,73 . Molecular docking and spectral analyzes indicate the interaction of AFM 1 with DNA. All these interactions show that the high frequency of CAs and MN formations resulting from AFM 1 application may result from interaction with DNA and the mechanism of genotoxicity can be explained by this interaction. DNA-AFM 1 interaction can lead to  Analysis of cytotoxic effects. The effects of AFM 1 and t-rsv on MI, which is an indicator of cell proliferation, are given in Fig. 9. MI levels in the control group, 10 mg/kg, 20 mg/kg t-rsv-treated groups were in the range of 13.9-14.3% and there was no statistical difference (p > 0.05). AFM 1 application caused a decrease in MI rate by reducing the number of dividing cells. 16 mg/kg AFM 1 application caused a decrease in MI rate of 32.5% compared to the control group. It was determined that MI rates improved in Group V and Group VI, which were treated with t-rsv + AFM 1 . This improvement was especially more pronounced in Group VI administered with 20 mg/kg t-rsv + AFM 1 , and MI increased 38.9% compared to Group IV treated with only AFM 1 . This healing feature of t-rsv can be explained by its regulatory role on the cell cycle. Macar et al. 74 reported that resveratrol administration has an increasing effect on the deteriorated MI in frequently dividing meristematic cells.

Macromolecule Free energy of binding (kcal/mol) Inhibition constant (Ki) Hydrogen bond interactions Hydrophobic interactions
Molecular docking of AFM 1 and tubulins. AFM 1 application caused a decrease in MI rates by reducing cell proliferation in bone marrow cells. These reductions are associated with the cytotoxic effects of AFM 1 . The   www.nature.com/scientificreports/ aneugenic effect of AFM 1 causes spindle damage, delays and disruptions in mitosis, and these delays reduce MI rates. AFM 1 can exhibit cytotoxic effects in many ways. In particular, the induction of large-scale MN formations by AFM 1 (Fig. 4), indicating an aneugenic effect, suggests possible damage to the spindle fibers. From this point, tubulin proteins in the structure of spindle fibers and AFM 1 docking were examined and the results are given in Fig. 10 and Table 5. AFM 1 formed hydrogen bondings with the Gln11, Ala12, Asn101, Ser140, Thr179, Phe141 and Ile171 residues of the tubulin alpha-1B chain, as well as hydrophobic interactions with the Ala180, Ile171 and Ala12 residues. AFM 1 formed hydrogen bondings and hydrophobic interactions with different aminoacid residues of tubulin beta chain with a binding energy of − 7.08 kcal/mol and an inhibition constant of 6.44 µM. The interaction between AFM 1 and tubulin proteins can cause conformational changes in protein structure and loss of function. The γ-,α-,β-tubulin heterodimers polymerize to form microtubules and the microtubules form the spindle. The spindle apparatus, consisting of hundreds of proteins, functions in the separation of sister chromatids during cell division 75,76 . Potential AFM 1 -tubulin interaction detected by molecular docking can cause disruption of the three-dimensional structure and inhibition of microtubule polymerization. This inhibition restricts the movement of chromosomes to the poles, resulting in both disruption in mitosis and formation of CA and MN. Although there is no data in the literature on AFM1-spindle interaction, many mycotoxins have been shown to cause abnormal spindle morphology and failure of spindle formation 77 .
Recovery effects of t-rsv. The reducing effects of t-rsv on the toxicity induced by AFM 1 are summarized in Fig. 11. The toxicity-reducing effect of t-rsv increased in parallel with the dose increase. 10 mg/kg t-rsv and 20 mg/kg t-rsv provided protection against abnormalities in serum parameters in the range of 14.66-34.26% and 45.69-60.3%, respectively. The genotoxic and cytotoxic effects of AFM 1 were determined by examining the MI Figure 10. The molecular docking of AFM 1 with tubulin proteins (a: α-tubulin, b: β-tubulin). www.nature.com/scientificreports/ rate, formation MN and CAs, and 20 mg/kg t-rsv reduced the frequency of MN by 48.9% and improved the rate of MI by 80.7%. 10 mg/kg and 20 mg/kg t-rsv treatment with AFM 1 resulted in a 22.6-63.9% reduction in CAs types. The most significant reduction was observed in gap formation, and 20 mg/kg t-rsv application reduced gap formation by 63.9%. There are many studies in the literature on the protective properties of resveratrol. However, in some studies, it is reported that resveratrol is cytotoxic 78 , and in some studies it has no toxic effect even at high doses 79 . In this study, 10 mg/kg and 20 mg/kg doses of resveratrol were tested, and it was determined that it did not have a toxic effect at these doses and showed a protective feature against AFM 1 toxicity. Figure 12a shows the correlation analysis of all parameters. Positive correlations are denoted by the color blue, whereas negative correlations are represented by the color red. Correlation coefficients are related to the color intensity and circle size. CAs, DNA Damage, buccal mucosa MN, erythrocyte MN, leukocyte MN, MDA liver, MDA kidney, ALT, AST, BUN and creatinine levels all showed a positive correlation with AFM 1 , but weight gain, MI rate, feed consumption, kidney weight, liver weight, GSH liver and GSH kidney levels all showed a negative correlation. T-resv was shown to have a positive correlation with weight gain, MI rate, feed consumption, kidney weight, liver weight, GSH liver and GSH kidney levels, but a negative correlation with CAs, DNA damage, buccal mucosa MN, erythrocyte MN, leukocyte MN, MDA liver, MDA kidney, ALT, AST, BUN, and creatinine levels, showing that it has a protective impact. Principal component analysis (PCA) was used to visualize the overall physiological, biochemical, and genetic impacts of AFM1 and t-rsv treatments on Swiss albino mice, as well as clustering amongst biomarkers after the application period and are given in Fig. 12b. PCA was used to visualize the overall physiological, biochemical, and genetic impacts of AFM1 and t-rsv treatments on Swiss albino mice, as well as clustering amongst biomarkers after the application period. Analysis of different toxicity biomarkers has provided a more reliable and more comprehensive view of the toxicity status and the interrelationships of these parameters. To minimize the complexity of data interpretation of multiple biomarker analysis, the current research used the statistical data-reduction tool PCA. In the current study, PCA analyzes of 4 physiological, 4 organ biochemistry, 4 blood biochemistry and 6 genetic parameters were analyzed and their relationships were examined. In Fig. 12b-1, which deals with PCA analyzes of physiological parameters, the first two dimensions of the biplot explained 89.4% of the overall variance, with the first axis (dim1) distinguishing control and treatment groups clearly (77.6%). The dim2 as a visualization aid accounted for 11.8% of the overall variance. As a result of the analysis, it was found that kidney weight, liver weight and feed consumption levels were close to each other, with a very positive component on the dim1 axis and a slight negative on the dim2 axis. Weight gain was on the positive axis of dim2 and dim1. PCA analyzes of organ biochemistry parameters are given in Fig. 12b-2. In the biplot, the first two dimensions, the first axis (dim1) 96.4% and the second axis (dim2) 2.8%, explain 99.2% of the overall variance. As a result of the analysis, it was found that GSH liver and GSH kidney levels were close to each other, with a very positive component on the dim1 axis and a slight positive on the dim2 axis. MDA liver and MDA kidney levels were found to be very negative on the dim1 axis and slightly positive on the dim2 axis. MDA liver levels were moderately positive in the dim2 axis, while MDA kidney levels were mildly negative. Fig. 12b-3 shows PCA analyses of blood biochemistry parameters. The first two dimensions in the biplot, the first axis (dim1) 96.3% and the second axis (dim2) 2.3%, explain 98.6% total variation. As an outcome of the assessment, it was found that creatinine, ALT, AST and BUN levels were very positive components on dim1 axis. It was determined that the levels of ALT, AST and BUN levels were close to each other, slightly negative in the dim2 axis and creatinine level was a slightly positive component in the dim2 axis. PCA analyzes of genetic parameters are given in Fig. 12b-4. The first two dimensions in the biplot, the first axis (dim1) 90.4% and the second axis (dim2) 6.7%, explain 97.1% total variance. As a result of the analysis, it was observed that the MI ratio had a very negative component in the dim1 axis and a positive component above the moderate level in the dim 2 axis. Buccal mucosa MN, erythrocyte MN, leukocyte MN, CAs and DNA Damage frequencies were found to be very positive components and very close to each other in the dim1 axis. These parameters are on the positive side of the dim2 axis and close to the axis. All these PCA analyzes confirm the interrelationships of the investigated parameters with each other.

Conclusion
Among mycotoxins, aflatoxins are the most effective on higher organisms, and it is very difficult to classify the effects of aflatoxins clearly since direct studies on humans cannot be conducted. Although there are many studies on aflatoxin species with chronic and acute effects in animals, studies on AFM 1 are generally related to the detection of its presence in milk and dairy products. In this study, the potentially toxic effects of AFM 1 in albino mice and the toxicity limiting property of t-rsv against these toxic effects were investigated. AFM 1 , which causes significant changes in selected physiological parameters, liver and kidney markers in albino mice, is an agent with cytotoxic and genotoxic effects and disrupts the antioxidant/oxidant balance. AFM 1 exhibited cytotoxic and genotoxic effects, respectively, by interacting with tubulin proteins, which are involved in cell division, and histone proteins, which have an important role in packaging and maintaining the integrity of DNA. With this study, the first data entry was provided to the literature regarding the formation MN in the buccal epithelium, leukocyte and erythrocyte induced by AFM 1 .
There are much data in the literature regarding the protective properties of resveratrol, which has antioxidant properties. However, there is no study reporting the protective effect of t-rsv against toxicity induced by AFM 1 . In this regard, this study is the first to report that t-rsv exhibits a dose-dependent protective role against AFM 1 toxicity. In the sustainability of high quality of life, it is very important to clarify the toxic effects of chemicals that contaminate organisms and to conduct research to reduce these effects. Studies investigating the toxic effects of chemicals and the toxicity-reducing role of natural products against these effects and elucidating the